The present invention relates to DNA molecules encoding enzymes which are involved in the starch synthesis of plants. These, enzymes represent two different isotypes of the soluble starch synthase as well as a starch granule-bound starch synthase. This invention furthermore relates to vectors, bacteria, as well as to plant cells transformed with the DNA molecules described and to plants regenerated from them.
Also, processes for the production of transgenic plants are described which, due to the introduction of DNA molecules encoding soluble or starch granule-bound starch synthases, synthesize a starch which is modified as regards its properties.
With respect to its increasing significance which has recently been ascribed to vegetal substances as regenerative sources of raw materials, one of the objects of biotechnological research is to try to adapt vegetal raw materials to the demands of the processing industry. In order to enable the use of modified regenerative raw materials in as many areas as possible, it is furthermore important to obtain a large variety of substances. Apart from oils, fats and proteins, polysaccharides constitute the essential regenerative raw materials derived from plants. Apart from cellulose, starch maintains an important position among the polysaccharides, being one of the most significant storage substances in higher plants. Besides maize, rice and wheat, potato plays an important role as starch producer.
The polysaccharide starch is a polymer made up of chemically homogeneous basic components, namely the glucose molecules. However, it constitutes a highly complex mixture from various types of molecules which differ from each other in their degree of polymerization and in the degree of branching of the glucose chains. Therefore, starch is not a homogeneous raw material. One differentiates particularly between amylose-starch, a basically non-branched polymer made up of xcex1-1,4-glycosidically branched glucose-molecules, and amylopectin-starch which in turn is a complex mixture of various branched glucose chains. The branching results from additional xcex1-1,6-glycosidic interlinkings. In plants which are typically used for starch production, such as, e.g., maize or potato, the synthesized starch consists of about 25% of amylose starch and of about 75% of amylopectin starch.
In order to enable as wide a use of starch as possible, it seems to be desirable that plants be provided which are capable of synthesizing modified starch which is particularly suitable for various uses. A possibility of providing such plants isxe2x80x94apart from breedingxe2x80x94in the specific genetic modification of the starch metabolism of starch-producing plants by means of recombinant DNA techniques. However, a prerequisite therefor is to identify and to characterize the enzymes involved in the starch synthesis and/or the starch modification as well as to isolate the respective DNA molecules encoding these enzymes.
The biochemical pathways which lead to the production of starch are basically known. The starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissues these are the chloroplasts, in photosynthetically inactive, starch-storing tissues the amyloplasts.
The most important enzymes involved in starch synthesis are starch synthases as well as branching enzymes. In the case of starch synthases various isotypes are described which all catalyze a polymerization reaction by transferring a glucosyl residue of ADP-glucose to xcex1-1,4-glucans. Branching enzymes catalyze the introduction of xcex1-1,6 branchings into linear xcex1-1,4-glucans.
Furthermore, it is discussed that other enzyme activities, such as hydrolytic or phosphorolytic activities, are involved in the synthesis of starch (Preiss in Oxford Survey of Plant Molecular and Cell Biology, Oxford University Press, Vol. 7 (1991), 59-114). It can furthermore not be precluded that the xe2x80x9cR enzymexe2x80x9d, or the so-called disproportionizing enzyme, and the starch phosphorylases also are involved in starch synthesis, although these enzymes so far have been connected with the degradation of starch.
Starch synthases may be divided up in two groups: the granule-bound starch syntheses (GBSS), which are mainly present bound to starch granules but also in soluble form, and the soluble starch synthases (SSS). Within these classifications, various isotypes are described for various species of plants. These isotypes differ from each other in their dependency on primer molecules (so-called xe2x80x9cprimer dependentxe2x80x9d (type II) and xe2x80x9cprimer independentxe2x80x9d (type I) starch synthases).
So far only in the case of the isotype GBSS I its exact function during starch synthesis has been successfully determined. Plants in which this enzyme activity has been strongly or completely reduced, synthesize starch free of amylose (a so-called xe2x80x9cwaxyxe2x80x9d starch) (Shure et al., Cell 35 (1983), 225-233; Visser et al., Mol. Gen. Genet. 225 (1991), 289-296; WO 92/11376); therefore this enzyme has been assigned a decisive role in synthesizing amylose-starch. This phenomenon is also observed in the cells of the green alga Chlamydomonas reinhardtii (Delrue et al., J. Bacteriol. 174 (1992), 3612-3620). In the case of Chlamydomonas it was furthermore demonstrated that GBSS I is not only involved in the synthesis of amylose but also has a certain influence on amylopectin synthesis. In mutants which do not show any GBSS I activity a certain fraction of the normally synthesized amylopectin, exhibiting long chain glucans, is missing.
The functions of the other isotypes of the granule-bound starch synthases, particularly GBSS II, and of the soluble starch synthases are so far not clear. It is assumed that soluble starch synthases, together with branching enzymes, are involved in the synthesis of amylopectin (see, e.g., Ponstein et al., Plant Physiol. 92 (1990), 234-241) and that they play an important role in the regulation of starch synthesis rate.
For potato, the isotypes GBSS I, GBSS II, as well as two or three isotypes of the soluble starch synthases, which so far have not been characterized further, have been identified (Ponstein et al. Plant Physiol. 92 (1990), 234-241; Smith et al., Planta 182 (1990), 599-604; Hawker et al., Phytochemistry 11 (1972), 1287-1293). Also for pea a GBSS II could be found (Dry et al., The Plant Journal 2,2 (1992), 193-202).
A cDNA encoding GBSS I from potato as well as a genomic DNA have already been described (Visser et al., Plant Sci. 64 (1989), 185-192; van der Leij et al., Mol. Gen. Genet. 228 (1991), 240-248). So far, no nucleic acid sequences encoding further granule-bound starch synthases or one of the soluble starch synthase isotypes from potato, have been reported.
Soluble starch synthases have been identified in several other plant species apart from potato. Soluble starch synthases have for example been isolated in homogeneous form from pea (Denyer and Smith, Planta 186 (1992), 609-617) and maize (WO 94/09144). In the case of pea it was found that the isotype of the soluble starch synthase identified as SSS II is identical with the granule-bound starch synthase GBSS II (Denyer et al., Plant J. 4 (1993), 191-198). In the case of other plant species the existence of several SSS-isotypes was described by means of chromatographic methods, as for example in the case of barley (Tyynelxc3xa4 and Schulman, Physiologia Plantarum 89 (1993) 835-841; Kreis, Planta 148 (1980), 412-416), maize (Pollock and Preiss, Arch. Biochem. Biophys. 204 (1980), 578-588) and wheat (Rijven, Plant Physiol. 81 (1986), 448-453). However, DNA sequences encoding these proteins have so far not been described.
A cDNA encoding a soluble starch synthase so far has only been described for rice (Baba et al., Plant Physiol. 103 (1993), 565-573).
In order to provide possibilities for modifying any desired starch-storing plant in such a way that they will synthesize a modified starch, respective DNA sequences encoding the various isotypes of granule-bound or soluble starch synthases have to be identified.
Therefore, it was the object of the present invention to provide DNA moleculesxe2x80x94especially from potatoxe2x80x94encoding enzymes involved in starch biosynthesis and by means of which genetically modified plants may be produced that show an elevated or reduced activity of those enzymes, thereby prompting a modification in the chemical and/or physical properties of the starch synthesized in these plants.
This object has been achieved by the provision of the embodiments described in the claims.
The invention therefore relates to DNA molecules encoding starch synthases, particularly such DNA molecules encoding the granule-bound starch synthases of the isotype II, as well as DNA molecules encoding soluble starch synthases.
The present invention particularly relates to DNA molecules encoding proteins with the biological activity of a granule-bound starch synthase of the isotype II (GBSSII) or a biologically active fragment of such a protein, such molecules preferably encoding proteins having the amino acid sequence indicated under Seq ID No. 8. Particularly, the invention relates to DNA molecules having the nucleotide sequence indicated under Seq ID No. 7, preferably molecules comprising the coding region indicated under Seq ID No. 7.
The subject matter of the invention are also DNA molecules encoding a GBSSII and the sequence of which differs from the nucleotide sequences of the above-described DNA molecules due to the degeneracy of the genetic code.
Furthermore, the invention relates to DNA molecules encoding GBSSII and hybridizing to any of the above-described DNA molecules. Such DNA molecules preferably are derived from starch-storing plants, particularly from dicotyledonous plants, and particularly preferred from potato.
The GBSSII proteins encoded by the DNA molecules according to the invention preferably have a molecular weight of 85xc2x15 kD. GBSSII proteins are mainly present bound to starch granules, however, they may also be present in soluble form.
Furthermore, the invention relates to DNA molecules encoding proteins with the biological activity of a soluble starch synthase of the isotype B (SSSB) or a biologically active fragment of such a protein, with such molecules preferably encoding proteins having the amino acid sequence indicated under Seq ID No. 10. In particular, the invention relates to DNA molecules having the nucleotide sequence indicated under Seq ID No. 9, preferably molecules comprising the coding region indicated under Seq ID No. 9.
Another subject matter of the invention are DNA molecules encoding an SSSB and the sequence of which differs from the nucleotide sequences of the above-described DNA molecules due to the degeneracy of the genetic code.
Furthermore, the invention relates to DNA molecules encoding SSSB and hybridizing to any of the above-described DNA molecules. An exception are the DNA molecules from rice. The SSSB proteins encoded by the DNA molecules according to the invention preferably have a molecular weight of 78xc2x15 kD.
The enzymatic properties of the SSSB proteins are described in the examples.
The invention furthermore relates to DNA molecules encoding proteins with the biological activity of a soluble starch synthase of the isotype A (SSSA). Such proteins can, for example, be characterized in that they are recognized by an antibody that is directed to the peptide having the amino acid sequence
NH2-GTGGLRDTVENC-COOH (Seq ID No. 13).
The enzymatic properties of the SSSA proteins are described in the examples.
An example of a DNA molecule encoding such a protein is a DNA molecule having the coding region depicted under Seq ID No. 11. This DNA molecule may be used to isolate from other organisms, in particular plants, DNA molecules encoding the SSSA proteins.
Thus, the present invention also relates to DNA molecules encoding proteins with the biological activity of a soluble starch synthase of the isotype A (SSSA) or a biologically active fragment of such a protein, with such molecules preferably encoding proteins having the amino acid sequence indicated under Seq ID No. 12. The invention particularly relates to DNA molecules having the nucleotide sequence indicated under Seq ID No. 11, preferably molecules comprising the coding region indicated under Seq ID No. 11.
Another subject matter of the invention are DNA molecules encoding SSSA and the sequence of which differs from the nucleotide sequences of the above-described DNA molecules due to a degeneracy of the genetic code.
Furthermore, the present invention relates to DNA molecules encoding SSSA and hybridizing to any of the above-described DNA molecules.
The SSSA protein preferably has an apparent molecular weight of about 120 to 140 kD, particularly of about 135 kD, in SDS gel electrophoresis.
In this invention the term xe2x80x9chybridizationxe2x80x9d signifies hybridization under conventional hybridizing conditions, preferably under stringent conditions as described for example in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). DNA molecules hybridizing to the DNA molecules according to the invention can basically be derived from any organism (i.e., prokaryotes or eukaryotes, particularly from bacteria, fungi, algae, plants or animal organisms) which possesses such DNA molecules. Preferably, they originate from monocotyledonous or dicotyledonous plants, in particular from useful plants, and particularly preferred from starch-storing plants.
DNA molecules hybridizing to the molecules according to the invention may be isolated, e.g., from genomic or from cDNA libraries from various organisms.
The identification and isolation of such DNA molecules from plants or other organisms may take place by using the DNA molecules according to the invention or parts of these DNA molecules or, as the case may be, the reverse complement strands of these molecules, e.g., by hybridization according to standard methods (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
As a probe for hybridization, e.g., DNA molecules may be used which exactly or basically contain the nucleotide sequences indicated under Seq ID No. 7, 9 or 11 or parts thereof. The fragments used as hybridization probe may also be synthetic DNA fragments which were produced by means of the conventional DNA synthesizing methods and the sequence of which is basically identical with that of a DNA molecule according to the invention. After identifying and isolating the genes hybridizing to the DNA sequences according to the invention, the sequence has to be determined and the properties of the proteins encoded by this sequence have to be analyzed.
The molecules hybridizing to the DNA molecules of the invention also comprise fragments, derivatives and allelic variants of the above-described DNA molecules which encode one of the proteins described above. Thereby, fragments are defined as parts of the DNA molecules, which are long enough in order to encode one of the described proteins. In this context, the term derivatives means that the DNA sequences of these molecules differ from the sequences of the above-mentioned DNA molecules at one or more positions and that they exhibit a high degree of homology to these DNA sequences. Hereby, homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably of more than 80% and still more preferably a sequence identity of more than 90%. The deviations occurring when comparing with the above-described DNA molecules might have been caused by deletion, substitution, insertion or recombination. Moreover, homology means that functional and/or structural equivalence exists between the respective DNA molecules or the proteins they encode. The DNA molecules, which are homologous to the above-described DNA molecules and represent derivatives of these DNA molecules, are generally variations of these molecules, that constitute modifications which exert the same biological function. These variations may be naturally occurring variations, for example sequences derived from other organisms, or mutations, whereby these mutations may have occurred naturally or they may have been introduced by means of a specific mutagenesis. Moreover, the variations may be synthetically produced sequences. The allelic variants may be naturally occurring as well as synthetically produced variants or variants produced by recombinant DNA techniques.
The proteins encoded by the various variants of the DNA molecules according to the invention exhibit certain common characteristics. Enzyme activity, molecular weight, immunologic reactivity, conformation etc. may belong to these characteristics as well as physical properties such as the mobility in gel electrophoresis, chromatographic characteristics, sedimentation coefficients, solubility, spectroscopic properties, stability; pH-optimum, temperature-optimum etc.
Significant characteristics of a starch synthase are: i) their localization within the stroma of the plastids of plant cells; ii) their capability of synthesizing linear xcex1-1,4-linked polyglucans using ADP-glucose as substrate. This activity can be determined as shown in Denyer and Smith (Planta 186 (1992), 606-617) or as described in the examples.
The DNA molecules according to the invention may basically originate from any organism expressing the proteins described, preferably from plants, particularly from starch-synthesizing or starch-storing plants. These plants may be monocotyledonous but also dicotyledonous plants. Particularly preferred are, e.g., cereals (such as barley, rye, oats, wheat, etc.), maize, rice, pea, cassava, potato, etc.
Furthermore, the invention relates to vectors, especially plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the above-mentioned DNA molecules of the invention.
In a preferred embodiment the DNA molecules contained in the vectors are linked to DNA elements that ensure the transcription and synthesis of a translatable RNA in prokaryotic and eukaryotic cells.
The expression of the DNA molecules of the invention in prokaryotic cells, e.g., in Escherichia coli, is interesting insofar as this enables a more precise characterization of the enzymatic activities of the enzymes encoding these molecules. In particular, it is possible to characterize the product being synthesized by the respective enzymes in the absence of other enzymes which are involved in the starch synthesis of the plant cell. This makes it possible to draw conclusions about the function, which the respective protein exerts during the starch synthesis within the plant cell.
Moreover, it is possible to introduce various mutations into the DNA molecules of the invention by means of conventional molecular-biological techniques (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), whereby the synthesis of proteins with possibly modified biological properties is induced. By means of this it is on the one hand possible to produce deletion mutants, in which DNA molecules are produced by continuing deletions at the 5xe2x80x2- or the 3xe2x80x2-end of the encoding DNA-sequence. These DNA molecules may lead to the synthesis of correspondingly shortened proteins. Such deletions at the 5xe2x80x2-end of the nucleotide sequence make it possible, for example, to identify amino acid sequences which are responsible for the translocation of the enzyme in the plastids (transit peptides). This allows for the specific production of enzymes which due to the removal of the respective sequences are no longer located in the plastids but within the cytosol, or which due to the addition of other signal sequences are located in other compartments.
On the other hand, point mutations might also be introduced at positions where a modification of the amino acid sequence influences, for example, the enzyme activity or the regulation of the enzyme. In this way, e.g., mutants with a modified Km-value may be produced, or mutants which are no longer subject to the regulation mechanisms by allosteric regulation or covalent modification usually occurring in cells.
Furthermore, mutants may be produced exhibiting a modified substrate or product specificity such as mutants that use ADP-glucose-6-phosphate instead of ADP-glucose as substrate. Moreover, mutants with a modified activity-temperature-profile may be produced.
For the genetic manipulation in prokaryotic cells the DNA molecules of the invention or parts of these molecules may be integrated into plasmids which allow for a mutagenesis or a sequence modification by recombination of DNA sequences. By means of standard methods (cf. Sambrook et al., 1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA) base exchanges may be carried out or natural or synthetic sequences may be added. In order to connect the DNA fragments, adapters or linkers may be attached to the fragments. Moreover, use can be made of manipulations which offer suitable restriction sites or which remove superfluous DNA or restriction sites. Wherever use is made of inserts, deletions or substitutions, in vitro mutagenesis, xe2x80x9cprimer repairxe2x80x9d, restriction or ligation may be used. For analyzing use is usually made of a sequence analysis, a restriction analysis or further biochemico-molecularbiological methods.
In a further embodiment the invention relates to host cells, in particular prokaryotic or eukaryotic cells, which contain a DNA molecule of the invention as described above or a vector of the invention. These are preferably bacterial cells or plant cells.
Furthermore, the proteins encoded by the DNA molecules of the invention are the subject-matter of the invention as well as methods for their production whereby a host cell of the invention is cultivated under conditions that allow for a synthesis of the protein and whereby the protein is then isolated from the cultivated cells and/or the culture medium.
It was found that by making available the nucleic acid molecules of the invention it is now possiblexe2x80x94by means of recombinant DNA techniquesxe2x80x94to interfere with the starch metabolism of plants in a way so far impossible and to modify it in such a way that a starch is synthesized which, e.g., is modified, compared to the starch synthesized in wild-type plants, with respect to its physico-chemical properties, especially the amylose/amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the pastification behavior, the size and/or the shape of the starch granule. Soluble starch synthases, play, e.g., a central role in the regulation of the synthesis rate of starch. There is the possibility of increasing the yield of genetically modified plants by increasing the activity of these enzymes or by making mutants available which are no longer subject to cell-specific regulation schemes and/or different temperature-dependencies with respect to their activity. The economic significance of the chance to interfere with the starch synthesis, namely of potato plants, is obvious: In Europe, for example, potato is one of the most important plants for producing starch apart from maize and wheat. About 20% of the starch produced in Europe per year is obtained from potatoes. Furthermore, potato starch exhibits some advantageous properties as compared to starch from maize or wheat, such as, e.g., a low protein and lipid content as well as relatively large starch granules and phosphate content. Therefore, if possible, potato starch is preferably used.
Therefore, it is possible to express the DNA molecules of the invention in plant cells in order to increase the activity of one or more starch synthases. Furthermore, the DNA molecules of the invention may be modified by means of methods known to the skilled person, in order to produce starch synthases which are no longer subject to the cell-specific regulation mechanisms or show modified temperature-dependencies or substrate or product specificities.
The synthesized protein may in principle be located in any desired compartment within the plant cell. In order to locate it within a specific compartment, the sequence ensuring the localization in the plastids must be deleted and the remaining coding regions optionally have to be linked to DNA sequences which ensure localization in the respective compartment. Such sequences are known (see, e.g., Braun et al., 1992, EMBO J. 11:3219-3227; Wolter et al., 1988, Proc. Natl. Acad. Sci. USA 85: 846-850; Sonnewald et al., 1991, Plant J. 1:95-106).
Thus, the present invention also relates to transgenic plant cells containing a DNA molecule of the invention, this DNA molecule being linked to regulatory DNA elements, which ensure the transcription in plant cells, especially with a promoter which is heterologous with respect to the DNA molecule.
By means of methods known to the skilled person the transgenic plant cells can be regenerated to whole plants. Thus, the plants obtained by regenerating the transgenic plant cells of the invention are also the subject-matter of the present invention. A further subject-matter of the invention are plants which contain the above-described transgenic plant cells. The transgenic plants may in principle be plants of any desired species, i.e., they may be monocotyledonous as well as dicotyledonous plants. These are preferably useful plants, such as cereals (rye, barley, oats, wheat etc.), rice, maize, peas, cassava or potatoes. The invention also relates to propagation material of the plants of the invention, e.g., fruits, seeds, tubers, cuttings etc.
Due to the expression or, as the case may be, additional expression of a DNA molecule of the invention, the transgenic plant cells and plants of the invention synthesize a starch which compared to starch synthesized in wild-type plants, i.e., non-transformed plants, is modified, in particular with respect to the viscosity of aqueous solutions of this starch and/or the phosphate content. Thus, the starch derived from transgenic plant cells and plants according to the invention is the subject-matter of the present invention.
A further subject-matter of the invention are transgenic plant cells, in which the activity of a protein according to the invention is reduced when compared to non-transformed plants. It was found that plant cells exhibiting a reduced activity of a protein of the invention synthesize a starch having modified chemical and/or physical properties as compared to that of wild-type plant cells.
The production of plant cells with a reduced activity of a protein of the invention may for example be achieved by using the DNA molecules of the invention. Possibilities are the expression of a corresponding antisense-RNA, of a sense-RNA for achieving a cosupression effect or the expression of a correspondingly constructed ribozyme, which specifically cleaves transcripts encoding a protein of the invention.
Preferably, an antisense RNA is expressed to reduce the activity of a protein of the invention in plant cells.
For this purpose, a DNA molecule can be used which comprises the complete sequence encoding a protein of the invention, including possibly existing flanking sequences as well as DNA molecules, which only comprise parts of the encoding sequence whereby these parts have to be long enough in order to prompt an antisense-effect within the cells. Basically, sequences with a minimum length of 15 bp, preferably with a length of 100-500 bp and for an efficient antisense-inhibition, in particular sequences with a length of more than 500 bp may be used. Generally DNA-molecules are used which are shorter than 5000 bp, preferably sequences with a length of less than 2500 bp. Preferably, use is made of DNA molecules that are homologous with respect to the plant species to be transformed.
Use may also be made of DNA sequences which are highly homologous, but not completely identical to the sequences of the DNA molecules of the invention. The minimal homology should be more than about 65%. Preferably, use should be made of sequences with homologies between 95 and 100%.
The transgenic plant cells of the invention can be regenerated to whole plants by means of methods known to the skilled person. Thus, plants containing the transgenic plant cells of the invention are also the subject-matter of the present invention. These plants generally are plants of any species, i.e., monocotyledonous and dicotyledonous plant. Preferably these plants are useful plants, especially starch-storing plants such as cereals (rye, barley, oats, wheat, etc.), rice, maize, peas, cassava or potatoes. The invention also relates to propagation material of the plants of the invention, such as fruit, seeds, tubers, cuttings, etc.
Due to the reduction of the activity of one of the proteins of the invention, the transgenic plant cells and plants of the invention synthesize a starch which is modified, compared to the starch from non-transformed plant cells or plants, in its chemical and/or physical properties. This starch exhibits for example a modified viscosity of its aqueous solutions and/or a modified phosphate content.
Thus, starch derived from the above-mentioned transgenic plant cells and plants is also the subject-matter of the invention.
The starches of the invention may be modified according to techniques known to the skilled person; in unmodified as well as in modified form they are suitable for use in foodstuffs or non-foodstuffs.
Basically, the possibilities of uses of the starch can be subdivided into two major fields. One field comprises the hydrolysis products of starch which mainly include glucose and glucan components obtained by enzymatic or chemical processes. They serve as starting materials for further chemical modifications and processes such as fermentation. In this context, it might be of importance that the hydrolysis process can be carried out simply and inexpensively. Currently, it is carried out substantially enzymatically using amyloglucosidase. It is thinkable that costs might be reduced by using lower amounts of enzymes for hydrolysis due to changes in the starch structure, e.g., increased surface of the grain, improved digestibility due to less branching or a steric structure, which limits the accessibility for the used enzymes.
The other area in which starch is used due to its polymer structure as so-called native starch, can be subdivided into two further areas:
1. Use in Foodstuffs
Starch is a classic additive for various foodstuffs, in which it essentially serves the purpose of binding aqueous additives and/or causes an increased viscosity or an increased gel formation. Important characteristic properties are flowing and sorption behavior, swelling and pastification temperature, viscosity and thickening performance, solubility of the starch, transparency and paste structure, heat, shear and acid resistance, tendency to retrogradation, capability of film formation, resistance to freezing/thawing, digestibility as well as the capability of complex formation with, e.g., inorganic or organic ions.
2. Use in Non-foodstuffs
The other major field of application is the use of starch as an adjuvant in various production processes or as an additive in technical products. The major fields of application for the use of starch as an adjuvant are, first of all, the paper and cardboard industry. In this field, the starch is mainly used for retention (holding back solids), for sizing filler and fine particles, as solidifying substance and for dehydration. In addition, the advantageous properties of starch with regard to stiffness, hardness, sound, grip, gloss, smoothness, tear strength as well as the surfaces are utilized.
2.1 Paper and Cardboard Industry
Within the paper production process, a differentiation can be made between four fields of application, namely surface, coating, mass and spraying.
The requirements on starch with regard to surface treatment are essentially a high degree of brightness, corresponding viscosity, high viscosity stability, good film formation as well as low formation of dust. When used in coating the solid content, a corresponding viscosity, a high capability to bind as well as a high pigment affinity play an important role. As an additive to the mass rapid, uniform, loss-free dispersion, high mechanical stability and complete retention in the paper pulp are of importance. When using the starch in spraying, corresponding content of solids, high viscosity as well as high capability to bind are also significant.
2.2 Adhesive Industry
A major field of application is, for instance, in the adhesive industry, where the fields of application are subdivided into four areas: the use as pure starch glue, the use in starch glues prepared with special chemicals, the use of starch as an additive to synthetic resins and polymer dispersions as well as the use of starches as extenders for synthetic adhesives. 90% of all starch-based adhesives are used in the production of corrugated board, paper sacks and bags, composite materials for paper and aluminum, boxes and wetting glue for envelopes, stamps, etc.
2.3 Textile and Textile Care Industry
Another possible use as adjuvant and additive is in the production of textiles and textile care products. Within the textile industry, a differentiation can be made between the following four fields of application: the use of starch as a sizing agent, i.e., as an adjuvant for smoothing and strengthening the burring behavior for the protection against tensile forces active in weaving as well as for the increase of wear resistance during weaving, as an agent for textile improvement mainly after quality-deteriorating pretreatments, such as bleaching, dying, etc., as a thickener in the production of dye pastes for the prevention of dye diffusion and as an additive for warping agents for sewing yarns.
2.4 Building Industry
The fourth area of application of starch is its use as an additive in building materials. One example is the production of gypsum plaster boards, in which the starch mixed in the thin plaster pastifies with the water, diffuses at the surface of the gypsum board and thus binds the cardboard to the board. Other fields of application are admixing it to plaster and mineral fibers. In ready-mixed concrete, starch may be used for the deceleration of the sizing process.
2.5 Ground Stabilization
Furthermore, the starch is advantageous for the production of means for ground stabilization used for the temporary protection of ground particles against water in artificial earth shifting. According to state-of-the-art knowledge, combination products consisting of starch and polymer emulsions can be considered to have the same erosion- and incrustation-reducing effect as the products used so far; however, they are considerably less expensive.
2.6 Use of Starch in Plant Protectives and Fertilizers
Another field of application is the use of starch in plant protectives for the modification of the specific properties of these preparations. For instance, starches are used for improving the wetting of plant protectives and fertilizers, for the dosed release of the active ingredients, for the conversion of liquid, volatile and/or odorous active ingredients into microcristalline, stable, deformable substances, for mixing incompatible compositions and for the prolongation of the duration of the effect due to a reduced disintegration.
2.7 Drugs, Medicine and Cosmetics Industry
Starch may also be used in the fields of drugs, medicine and in the cosmetics industry. In the pharmaceutical industry, the starch may be used as a binder for tablets or for the dilution of the binder in capsules. Furthermore, starch is suitable as disintegrant for tablets since, upon swallowing, it absorbs fluid and after a short time it swells so much that the active ingredient is released. For qualitative reasons, medicinal flowance and dusting powders are further fields of application. In the field of cosmetics, the starch may for example be used as a carrier of powder additives, such as scents and salicylic acid. A relatively extensive field of application for the starch is toothpaste.
2.8 Starch as an Additive in Coal and Briquettes
The use of starch as an additive in coal and briquettes is also thinkable. By adding starch, coal can be quantitatively agglomerated and/or briquetted in high quality, thus preventing premature disintegration of the briquettes.
Barbecue coal contains between 4 and 6% added starch, calorated coal between 0.1 and 0.5%. Furthermore, the starch is suitable as a binding agent since adding it to coal and briquette can considerably reduce the emission of toxic substances.
2.9 Processing of Ore and Coal Slurry
Furthermore, the starch may be used as a flocculant in the processing of ore and coal slurry.
2.10 Starch as an Additive in Casting
Another field of application is the use as an additive to process materials in casting. For various casting processes cores produced from sands mixed with binding agents are needed. Nowadays, the most commonly used binding agent is bentonite mixed with modified starches, mostly swelling starches.
The purpose of adding starch is increased flow resistance as well as improved binding strength. Moreover, swelling starches may fulfill more prerequisites for the production process, such as dispersability in cold water, rehydratisability, good mixability in sand and high capability of binding water.
2.11 Use of Starch in Rubber Industry
In the rubber industry starch may be used for improving the technical and optical quality. Reasons for this are improved surface gloss, grip and appearance. For this purpose, the starch is dispersed on the sticky rubberized surfaces of rubber substances before the cold vulcanization. It may also be used for improving the printability of rubber.
2.12 Production of Leather Substitutes
Another field of application for the modified starch is the production of leather substitutes.
2.13 Starch in Synthetic Polymers
In the plastics market the following fields of application are emerging: the integration of products derived from starch into the processing process (starch is only a filler, there is no direct bond between synthetic polymer and starch) or, alternatively, the integration of products derived from starch into the production of polymers (starch and polymer form a stable bond).
The use of the starch as a pure filler cannot compete with other substances such as talcum. This situation is different when the specific starch properties become effective and the property profile of the end products is thus clearly changed. One example is the use of starch products in the processing of thermoplastic materials, such as polyethylene. Thereby, starch and the synthetic polymer are combined in a ratio of 1:1 by means of coexpression to form a xe2x80x98master batchxe2x80x99, from which various products are produced by means of common techniques using granulated polyethylene. The integration of starch in polyethylene films may cause an increased substance permeability in hollow bodies, improved water vapor permeability, improved antistatic behavior, improved anti-block behavior as well as improved printability with aqueous dyes. Present disadvantages relate to insufficient transparency, reduced tensile strength as well as reduced extensibility.
Another possibility is the use of the starch in polyurethane foams. Due to the adaptation of starch derivatives as well as due to the optimization of processing techniques, it is possible to specifically control the reaction between synthetic polymers and the starch""s hydroxy groups. The results are polyurethane films having the following property profiles due to the use of starch: a reduced coefficient of thermal expansion, decreased shrinking behavior, improved pressure/tension behavior, increased water vapor permeability without a change in water acceptance, reduced flammability and cracking density, no drop off of combustible parts, no halides and reduced aging. Disadvantages that presently still exist are reduced pressure and impact strength.
Product development of film is not the only option. Also solid plastics products, such as pots, plates and bowls can be produced by means of a starch content of more than 50%. Furthermore, the starch/polymer mixtures offer the advantage that they are much easier biodegradable.
Furthermore, due to their extreme capability to bind water, starch graft polymers have gained utmost importance. These are products having a backbone of starch and a side lattice of a synthetic monomer grafted on according to the principle of radical chain mechanism. The starch graft polymers available nowadays are characterized by an improved binding and retaining capability of up to 1000 g water per g starch at a high viscosity. The fields of application of these super absorbers have extended over the last few years and they are used mainly in the hygiene field, e.g., in products such as diapers and sheets, as well as in the agricultural sector, e.g., in seed pellets.
What is decisive for the use of the new starch modified by recombinant DNA techniques are, on the one hand, structure, water content, protein content, lipid content, fiber content, ashes/phosphate content, amylose/amylopectin ratio, distribution of the relative molar mass, degree of branching, granule size and shape as well as crystallization, and on the other hand, the properties resulting in the following features: flow and sorption behavior, pastification temperature, viscosity, thickening performance, solubility, paste structure, transparency, heat, shear and acid resistance, tendency to retrogradation, capability of gel formation, resistance to freezing/thawing, capability of complex formation, iodine binding, film formation, adhesive strength, enzyme stability, digestibility and reactivity.
The production of modified starch by genetically operating with a transgenic plant may modify the properties of the starch obtained from the plant in such a way as to render further modifications by means of chemical or physical methods superfluous. On the other hand, the starches modified by means of recombinant DNA techniques might be subjected to further chemical modification, which will result in further improvement of the quality for certain of the above-described fields of application. These chemical modifications are principally known to the person skilled in the art. These are particularly modifications by means of
heat treatment
acid treatment
oxidation and
esterification
leading to the formation of phosphate, nitrate, sulfate, xanthate, acetate and citrate starches. Other organic acids may also be used for the esterification:
formation of starch ethers starch alkyl ether, O-allyl ether, hydroxylalkyl ether, O-carboxylmethyl ether, N-containing starch ethers, P-containing starch ethers and S-containing starch ethers.
formation of branched starches
formation of starch graft polymers.
In order to express the DNA molecules of the invention in sense- or antisense-orientation in plant cells, these are linked to regulatory DNA elements which ensure the transcription in plant cells. Such regulatory DNA elements are particularly promoters.
The promoter may be selected in such a way that the expression takes place constitutively or in a certain tissue, at a certain point of time of the plant development or at a point of time determined by external circumstances. With respect to the plant the promoter may be homologous or heterologous. A suitable promoter for a constitutive expression is, e.g., the 35S RNA promoter of the Cauliflower Mosaic Virus. For a tuber-specific expression in potatoes the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) or a promoter which ensures expression only in photosynthetically active tissues, e.g., the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) may be used. For an endosperm-specific expression the HMG promoter from wheat, or promoters from zein genes from maize are suitable.
Furthermore, a termination sequence may exist which serves to correctly end the transcription and to add a poly-A-tail to the transcript which is believed to stabilize the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged as desired.
According to the invention, it is basically possible to produce plants in which only the activity of one isotype of the SSS or the GBSS II is modified, and also plants in which the activities of several starch synthase forms are simultaneously modified. Thereby, all kinds of combinations and permutations are thinkable.
By modifying the activities of one or more isotypes of the starch synthases in plants, a synthesis of a starch modified in its structure is brought about.
By increasing the activity of one or more isotypes of the starch synthases in the cells of the starch-storing tissue of transformed plants such as in the potato tuber or in the endosperm of maize or wheat, increased yields may be the result.
Since the DNA sequence encoding the GBSS I from potato is already known (Visser et al., Plant Sci. 64 (1989), 185-192), DNA sequences encoding all starch synthases so far identified in potato are available. This allows for the identification of the function of the individual isotypes in the starch biosynthesis as well as for the production of genetically modified plants in which the activity of at least one of these enzymes is modified. This enables the synthesis of starch with a modified structure and therefore with modified physico-chemical properties in the plants manipulated in such a way.
The DNA molecules of the invention may be used in order to produce plants in which the activity of the starch synthases mentioned is elevated or reduced and in which at the same time the activities of other enzymes involved in the starch biosynthesis are modified. Thereby, all kinds of combinations and permutations are thinkable. For example, DNA molecules encoding the SSS proteins or GBSS II may be introduced into plant cells according to the process described above in which the synthesis of endogenous GBSS I-proteins is already inhibited due to an antisense-effect (as described in Visser et al., Mol. Gen. Genet. 225 (1991), 289-296),or in which the synthesis of the branching enzyme is inhibited (as described in WO92/14827).
If the inhibition of the synthesis of several starch synthases in transformed plants is to be achieved, DNA molecules can be used for transformation, which at the same time contain several regions in antisense-orientation controlled by a suitable promoter and encoding the corresponding starch synthases. Hereby, each sequence may be controlled by its own promoter or else the sequences may be transcribed as a fusion of a common promoter. The last alternative will generally be preferred as in this case the synthesis of the respective proteins should be inhibited to approximately the same extent.
Furthermore, it is possible to construct DNA molecules in which apart from DNA sequences encoding starch synthases other DNA sequences are present encoding other proteins involved in the starch synthesis or modification and coupled to a suitable promoter in antisense orientation. Hereby, the sequences may again be connected up in series and be transcribed by a common promoter. For the length of the individual coding regions used in such a c construct the above-mentioned facts concerning the production of antisense-construct are also true. There is no upper limit for the number of antisense fragments transcribed from a promoter in such a DNA molecule. The resulting transcript, however, should not be longer than 10 kb, preferably 5 kb.
Coding regions which are located in antisense-orientation behind a suitable promoter in such DNA molecules in combination with other coding regions, may be derived from DNA sequences encoding the following proteins: granule-bound starch synthases (GBSS I and II), other soluble starch synthases (SSS I and II), branching enzymes (KoBmann et al., Mol. Gen. Genet. 230 (1991) 39-44), debranching enzymes (R enzymes), disproportionizing enzymes (Takaha et al., J. Biol. Chem. 268 (1993), 1391-1396) and starch phosphorylases. This enumeration merely serves as an example. The use of other DNA sequences within the framework of such a combination is also thinkable.
By means of such constructs it is possible to inhibit the synthesis of several enzymes at the same time within the plant cells transformed with these molecules.
In order to prepare the integration of foreign genes into higher plants a high number of cloning vectors are at disposal, containing a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples for such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence may be integrated into the vector at a suitable restriction site. The obtained plasmid is used for the transformation of E. coli cells. Transformed E. coli cells are cultivated in a suitable medium end subsequently harvested and lysed. The plasmid is recovered. As an analyzing method for the characterization of the obtained plasmid DNA use is generally made of restriction analysis, gel electrophoresis and other biochemico-molecularbiological methods. After each manipulation the plasmid DNA may be cleaved and the obtained DNA fragments may be linked to other DNA sequences. Each plasmid DNA may be cloned into the same or in other plasmids.
In order to integrate DNA into plant host cells a wide range of techniques are at disposal. These techniques comprise the transformation of plant cells with T-DNA by using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation medium, the fusion of protoplasts, the injection and the electroporation of DNA, the integration of DNA by means of the biolistic method as well as further possibilities.
In the case of injection and electroporation of DNA into plant cells, there are no special demands made to the plasmids used. Simple plasmids such as pUC derivatives may be used. However, in case that whole plants are to be regenerated from cells transformed in such a way, a selectable marker gene should be present.
Depending on the method of integrating desired genes into the plant cell, further DNA sequences may be necessary. If the Ti- or Ri-plasmid is used, e.g., for the transformation of the plant cell, at least the right border, more frequently, however, the right and left border of the Ti- and Ri-plasmid T-DNA has to be connected to the foreign gene to be integrated as a flanking region.
If Agrobacteria are used for the transformation, the DNA which is to be integrated must be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. Due to sequences homologous to the sequences within the T-DNA, the intermediate vectors may be integrated into the Ti- or Ri-plasmid of the Agrobacterium due to homologous recombination. This also contains the vir-region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in Agrobacteria. By means of a helper plasmid the intermediate vector may be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors may replicate in E. coli as well as in Agrobacteria. They contain a selectable marker gene as well as a linker or polylinker which is framed by the right and the left T-DNA border region. They may be transformed directly into the Agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187). The Agrobacterium acting as host cell should contain a plasmid carrying a vir-region. The vir-region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The Agrobacterium transformed in such a way is used for the transformation of plant cells.
The use of T-DNA for the transformation of plant cells was investigated intensely and described sufficiently in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.
For transferring the DNA into the plant cells, plant explants may suitably be co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. pieces of leaves, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) whole plants may then be regenerated in a suitable medium which may contain antibiotics or biozides for the selection of transformed cells. The plants obtained in such a way may then be examined as to whether the integrated DNA is present or not. Other possibilities in order to integrate foreign DNA by using the biolistic method or by transforming protoplasts are known to the skilled person (cf., e.g., Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Puhler, P. Stadler, editors), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).
Once the introduced DNA has been integrated in the genome of the plant cell, it usually continues to be stable there and also remains within the descendants of the originally transformed cell. It usually contains a selectable marker which confers resistance against a biozide or against an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricine, etc. to the transformed plant cells. The individually selected marker should therefore allow for a selection of transformed cells to cells lacking the integrated DNA.
The transformed cells grow in the usual way within the plants (see also McCormick et al., 1986, Plant Cell Reports 5: 81-84).
The resulting plants can be cultivated in the usual way and cross-bred with plants having the same transformed genetic heritage or another genetic heritage. The resulting hybrid individuals have the corresponding phenotypic properties. Two or more generations should be grown in order to ensure whether the phenotypic feature is kept stably and whether it is transferred. Furthermore, seeds should be harvested in order to ensure that the corresponding phenotype or other properties will remain.
The plasmid pBinARHyg used in this invention was deposited with Deutsche Sammlung von Mikroorganismen (DSM) [German collection of microorganisms] in Brunswick, Federal Republic of Germany, as international recognized depositary authority in accordance with the stipulations of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on Jan. 20, 1994 under deposit no. DSM 9505.
Abbreviations used
bp base pair
GBSS granule-bound starch synthase
IPTG isopropyl xcex2-D-thiogalacto-pyranoside
SSS soluble starch synthase
PMSF phenylmethylsulfonylfluoride
VK full-length clone
Media and solutions used in the examples:
20xc3x97SSC
175.3 g NaCl
88.2 g sodium citrate
ad 1000 ml with ddH2O
pH 7.0 with 10 N NaOH
Buffer A
50 mM Tris-HCl pH 8.0
2.5 mM DTT
2 mM EDTA
0.4 mM PMSF
10% glycerol
0.1% sodium dithionite
Buffer B
50 mM Tris-HCl pH 7.6
2.5 mM DTT
2 mM EDTA
Buffer C
0.5 M sodium citrate pH 7.6
50 mM Tris-HCl pH 7.6
2.5 mM DTT
2 mM EDTA
10xc3x97TBS
0.2 M Tris-HCl pH 7.5
5.0 M NaCl
10xc3x97TBST
10xc3x97TBS
0.1% (vol./vol.) Tween 20
Elution buffer
25 mM Tris pH 8.3
250 mM glycine
Dialysis buffer
50 mM Tris-HCl pH 7.0
50 mM NaCl
2 mM EDTA
14.7 mM xcex2-mercaptoethanol
0.5 mM PMSF
Protein buffer
50 mM sodium phosphate buffer pH 7.2
10 mM EDTA
0.5 mM PMSF
14.7 mM xcex2-mercaptoethanol